Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation.

Autor: Fumian TM; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil. Electronic address: tuliomf@ioc.fiocruz.br., Leite JP; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil., Rocha MS; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil., de Andrade JS; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil., Fioretti JM; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil., de Assis RM; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil., Assis MR; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil., Fialho AM; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil., Miagostovich MP; Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Brazil.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2016 Feb; Vol. 228, pp. 123-9. Date of Electronic Publication: 2015 Nov 22.
DOI: 10.1016/j.jviromet.2015.11.008
Abstrakt: Rotavirus A (RVA) and noroviruses (NoV) are the major viral agents of acute gastroenteritis (AGE) worldwide. In the present study, we aimed to evaluate the performance of a one-step duplex quantitative RT-PCR (dRT-qPCR) assay, established for detection and quantification of RVA and NoV genogroup II (GII) using a single DNA standard curve (SC), as well as to investigate the association between fecal viral load and optical density (OD) values, and viruses' genotyping. The results obtained by dRT-qPCR in 530 fecal samples from AGE cases were compared with methods employed for the diagnosis of those viruses as follows: enzyme immunoassay (EIA) and polyacrylamide gel electrophoresis (PAGE) for RVA; and qualitative PCR for NoV. By using dRT-qPCR, we detected RVA and NoV in 353 (66%), increasing the positivity rate by 22.5% for RVA and 11.5% NoV, comparing the number of positive samples. RVA and NoV GII were detected in a range of 5.17 × 10(3) to 6.56 × 10(9) and 3.76 × 10(3) to 9.13 × 10(10) genome copies per gram of feces, respectively. We observed a significant direct correlation between genome copies values and optical density, using dRT-qPCR and EIA assays, respectively (Spearman ρ=0.41; p<0.0001). Viruses characterization demonstrated a predominance of NoV GII.4 Sidney 2012 variant during October 2013 to February 2014, followed by the emergence of RVA genotype G12P[8] in 2014. The established assay using a single SC provides an early feedback concerning detection and quantification, with the advantage of detecting simultaneously RVA and NoV GII, reducing time and reagent costs.
(Copyright © 2015 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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